Background: This study aimed to investigate the effects of matrine (MAT) on the drug resistance of K562/ ADM cells.Methods: K562/S and K562/ADM cells were treated with different concentrations of vincristine (VCR), adriamycin (ADM), and/or MAT for 48 h. The cell viability was determined by MTT assay, and the drugresistant multiple and drug-resistant reversal multiple were calculated based on half-inhibitory concentration (IC50). The cell apoptosis, as well as cell cycle were detected by flow cytometry. The autophagy was evaluated by transmission electron microscope and GFP-LC3 staining. Autophagy-related protein levels of LC3 II, and P62 were detected by Western blot.Results: MAT reduced the viability of K562/S and K562/ADM cells in a dose-dependent manner, and 0.5 mg/mL was identified as the non-cytotoxic concentration. MAT eliminated the drug resistance of K562/ ADM cells to ADM and VCR, and the drug resistance reversal multiple was 10.12 and 4.91, respectively. Chloroquine (CQ), a lysosomal inhibitor significantly reversed the inhibitory effect of MAT on the viability of K562/ADM cells. In addition, MAT induced the apoptosis of K562/ADM cells through arresting the cell cycle at G0/G1 phase in a dose-dependent manner. MAT also increased the autophagic vacuoles, the LC3+ punctate fluorescence, as well as the LC3 II protein level, and decreased P62 protein level in K562/ADM cells in a dose-dependent manner.Conclusions: MAT reversed the drug resistance of K562/ADM cells to ADM and VCR through promoting autophagy.